Methods for producing preparations of recombinant AAV virions substantially free of empty capsids

ABSTRACT

Methods for separating AAV empty capsids from mixtures of AAV vector particles and AAV empty capsids are described. The methods use column chromatography techniques and provide for commercially viable levels of recombinant AAV virions.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims the benefit under 35 U.S.C. §119(e) to U.S.Provisional Application Ser. Nos. 60/472,384, filed May 21, 2003 and60/549,756, filed Mar. 2, 2004, which applications are incorporatedherein by reference in their entireties.

TECHNICAL FIELD

The invention relates to methods for purifying adeno-associated virus(AAV) virions. More particularly, the invention relates to methods forpurifying recombinant AAV (rAAV) virions containing packaged genomesfrom mixtures of AAV virions containing both packaged rAAV virions andAAV empty capsids lacking said genomes.

BACKGROUND

Gene therapy methods are currently being developed that safely andpersistently deliver therapeutically effective quantities of geneproducts to patients. Using these methods, a nucleic acid molecule canbe introduced directly into a patient (in vivo gene therapy), or intocells isolated from a patient or a donor, which are then subsequentlyreturned to the patient (ex vivo gene therapy). The introduced nucleicacid then directs the patient's own cells or grafted cells to producethe desired therapeutic product. Gene therapy also allows clinicians toselect specific organs or cellular targets (e.g., muscle, blood cells,brain cells, etc.) for therapy.

Nucleic acids may be introduced into a patient's cells in several ways,including viral-mediated gene delivery, naked DNA delivery, andtransfection methods. Viral-mediated gene delivery has been used in amajority of gene therapy trials. C. P. Hodgson Biotechnology (1995)13:222-225. The recombinant viruses most commonly used are based onretrovirus, adenovirus, herpesvirus, pox virus, and adeno-associatedvirus (AAV).

Recombinant adeno-associated viral vectors hold promise as gene deliveryvectors for human gene therapy. However, one significant obstacle tousing such vectors as drugs is the development of a truly scaleableprocess to produce and purify the vector at commercially viable levels.For a review of the challenges involved in scaling AAV vector productionfor commercial use, see Qu and Wright, Cur. Opin. Drug Disc. andDevelop. (2000) 3:750-755. Recently, several potentially scalable columnchromatography techniques to purify rAAV virions have been developed.While these column chromatography-based purification methods havedemonstrated that rAAV virions can be purified at large scale, thepreparation of purified virions using column chromatography contains asignificant amount of AAV empty capsids. The typical ratio of emptycapsids to virions containing a heterologus gene of interest (“AAVvector particles”) is about 10 or higher, i.e., approximately 90% of therecovered vectors are empty capsids.

The presence of a large amount of empty capsids may hinder clinicalapplications, e.g., by eliciting unwanted immune responses to the capsidprotein or by competing for target cell surface binding sites.Consequently, techniques have been developed to remove the empty capsidsfrom rAAV virion preparations. These techniques typically rely onultracentrifugation, for example gradient centrifugation in cesiumchloride or iodixanol. Such centrifugation techniques are laborintensive, typically result in low vector yield, and are not scalable.Kaludov et al., (2002) Hum. Gene Ther. 13:1235-1243, describe methods ofpurifying rAAV-2, -4 and -5 vectors using anion exchange columns.However, the experimenters were only able to recover 2%, 0.6% and 6.3%,respectively, as packaged genomes, even after pooling the eluates andconcentrating the fractions.

Thus, there remains a need for new ways of eliminating or reducing thenumbers of empty capsids from stocks of AAV vector particles so thatmanufacturing capability is enhanced.

SUMMARY OF THE INVENTION

The present invention is based on the discovery of efficient andcommercially viable methods for preparing stocks of rAAV virions withreduced amounts of empty capsids. The inventors herein have found thatempty capsids can be separated from rAAV virions containing geneticmaterial (“AAV vector particles”) using column chromatographytechniques. This result is surprising as it was previously believed thatempty and packaged capsids had identical surface properties. To the bestof the inventors' knowledge, this is the first demonstration that viralparticle charge and/or the charge-density are different between emptyparticles and full particles. The techniques described herein provideefficient and scalable methods to separate AAV empty capsids from AAVvector particles.

Accordingly, in one embodiment, the invention is directed to a methodfor purifying AAV vector particles from an AAV preparation comprisingAAV vector particles and AAV empty capsids, to provide an AAV productsubstantially free of AAV empty capsids. The method comprises:

(a) providing a host cell comprising AAV vector particles;

(b) lysing the host cell to obtain a crude cell lysate comprising AAVvector particles and AAV empty capsids;

(c) applying the crude cell lysate to a first cation exchangechromatograhphy column under conditions whereby the AAV vector particlesand the AAV empty capsids bind the column;

(d) eluting the AAV vector particles and the AAV empty capsids undernon-separating conditions to provide an AAV preparation comprising AAVvector particles and AAV empty capsids;

(e) applying the AAV preparation from (d) to a second cation exchangechromatography column under conditions whereby the AAV vector particlesand the AAV empty capsids bind the column;

(f) adding a low salt buffer to the column from (e) under conditionswhereby AAV vector particles are eluted and AAV empty capsids remainbound to the column; and

(g) collecting eluted fractions from (f) that comprise AAV vectorparticles to provide an AAV product substantially free of AAV emptycapsids.

In additional embodiments, the above method further comprises:

(h) applying the fractions from step (g) to an anion exchangechromatography column under conditions whereby the AAV vector particlesand AAV empty capsids, if present, bind the column;

(i) adding a low salt buffer to the column from (h) under conditionswhereby AAV empty capsids are eluted and AAV vector particles remainbound to the column;

(j) adding a high salt buffer to the column from (i) under conditionswhereby AAV vector particles are eluted;

(k) collecting fractions from (j) that comprise AAV vector particles toprovide an AAV product substantially free of AAV empty capsids.

In yet a further embodiment, the invention is directed to a method forpurifying AAV vector particles from an AAV preparation comprising AAVvector particles and AAV empty capsids, to provide an AAV productsubstantially free of AAV empty capsids. The method comprises:

(a) providing a host cell comprising AAV vector particles;

(b) lysing the host cell to obtain a crude cell lysate comprising AAVvector particles and AAV empty capsids;

(c) clarifying the crude cell lysate to provide a clarified cell lysate;

(d) applying the clarified cell lysate to a first cation exchangechromatograhphy column comprising a matrix with the functional ligandR—SO₃—, under conditions whereby the AAV vector particles and the AAVempty capsids bind the column;

(e) eluting the AAV vector particles and the AAV empty capsids undernon-separating conditions to provide an AAV preparation comprising AAVvector particles and AAV empty capsids;

(f) applying the AAV preparation from (e) to a second cation exchangechromatography column under conditions whereby the AAV vector particlesand the AAV empty capsids bind the column;

(g) adding a low salt buffer to the column from (f) under conditionswhereby AAV vector particles are eluted and AAV empty capsids remainbound to the column;

(h) collecting eluted fractions from (g) that comprise AAV vectorparticles;

(i) applying the fractions from step (h) to an anion exchangechromatography column under conditions whereby said AAV vector particlesand AAV empty capsids, if present, bind the column;

(j) adding a low salt buffer to the column from (i) under conditionswhereby AAV empty capsids are eluted and AAV vector particles remainbound to the column;

(k) adding a high salt buffer to the column from (j) under conditionswhereby AAV vector particles are eluted; and

(l) collecting eluted fractions from (k) that comprise AAV vectorparticles to provide an AAV product substantially free of AAV emptycapsids.

In another embodiment, the invention is directed to a method forpurifying AAV vector particles from an AAV preparation comprising AAVvector particles and AAV empty capsids, to provide an AAV productsubstantially free of AAV empty capsids. The method comprises:

(a) providing a host cell comprising AAV vector particles;

(b) lysing the host cell to obtain a crude cell lysate comprising AAVvector particles and AAV empty capsids;

(c) applying the crude cell lysate to a cation exchange chromatograhphycolumn under conditions whereby the AAV vector particles and the AAVempty capsids bind the column;

(d) eluting the AAV vector particles and the AAV empty capsids undernon-separating conditions to provide an AAV preparation comprising AAVvector particles and AAV empty capsids;

(e) applying the AAV preparation from (d) to an anion exchangechromatography column under conditions whereby the AAV vector particlesand the AAV empty capsids bind the column;

(f) adding a low salt buffer to the column from (e) under conditionswhereby AAV empty capsids are eluted and AAV vector particles remainbound to the column;

(g) adding a high salt buffer to the column from (f) under conditionswhereby AAV vector particles are eluted;

(h) collecting eluted fractions from (g) that comprise AAV vectorparticles to provide an AAV product substantially free of AAV emptycapsids.

In additional embodiments, the above method further comprises:

(i) applying the AAV preparation from (h) to a second anion exchangechromatography column under conditions whereby the AAV vector particlesand the AAV empty capsids, if present, bind the column;

(j) adding a low salt buffer to the column from (i) under conditionswhereby AAV empty capsids are eluted and AAV vector particles remainbound to the column;

(k) adding a high salt buffer to the column from (j) under conditionswhereby AAV vector particles are eluted;

(l) collecting eluted fractions from (k) that comprise AAV vectorparticles to provide an AAV product substantially free of AAV emptycapsids.

In alternative embodiments, the method further comprises:

(i) applying the AAV preparation from (h) to a second cation exchangechromatography column under conditions whereby the AAV vector particlesand the AAV empty capsids bind the column;

(j) adding a low salt buffer to the column from (i) under conditionswhereby AAV vector particles are eluted and AAV empty capsids remainbound to the column; and

(k) collecting eluted fractions from (j) that comprise AAV vectorparticles to provide an AAV product substantially free of AAV emptycapsids.

In certain embodiments of all of the above methods, the first cationexchange column and/or the second cation exchange column comprises acarboxymethylated or sulfonated matrix, such as a matrix that comprisesthe functional ligand R—SO₃—.

In additional embodiments of all of the above methods, the AAV vectorparticles are present in the AAV product in an amount of at least 50%,such as in an amount of at least 75%, e.g. in an amount of at least 85%,or at least 90%.

In yet further embodiments of all of the above methods, the AAV vectorparticles are derived from AAV-2 or AAV-5.

These and other embodiments of the subject invention will readily occurto those of skill in the art in view of the disclosure herein.

BRIEF DESCRIPTION OF THE FIGURES

FIGS. 1A and 1B show the binding characteristics of a crude lysatecontaining both AAV vector particles and AAV empty capsids. In FIG. 1A,the resins tested were as follows: Lane 1: control; Lanes 2 and 3: MACROPREP Q (strong anion-exchanger available from BioRad, Hercules, Calif.);Lanes 4 and 5: UNOSPHERE Q (strong anion-exchanger available fromBioRad, Hercules, Calif.); Lanes 6 and 7: POROS 50HQ (stronganion-exchanger available from Applied Biosystems, Foster City, Calif.);Lanes 8 and 9: POROS 50D (weak anion-exchanger available from AppliedBiosystems, Foster City, Calif.). In FIG. 1B, the resins tested were asfollows: Lane 1: control; Lanes 2 and 3: POROS 50PI (weakanion-exchanger available from Applied Biosystems, Foster City, Calif.);Lanes 4 and 5: SOURCE 30Q (strong anion-exchanger available fromAmersham Biosciences, Piscataway, N.J.); Lanes 6 and 7: DEAE SEPHAROSE(weak anion-exchanger available from Amersham Biosciences, Piscataway,N.J.); Lanes 8 and 9: Q SEPHAROSE (strong anion-exchanger available fromAmersham Biosciences, Piscataway, N.J.). For both FIGS. 1A and 1B, Lanes2, 4, 6 and 8 used a low salt (50 mM NaCl) washing fraction; Lanes 3, 5,7 and 9 used a high salt (1M NaCl) washing fraction.

FIG. 2 shows an analysis of AAV empty capsids and AAV vector particles(Vgs) before and after separation using anion exchange chromatography asdescribed in the examples.

FIG. 3 is a depiction of a silver-stained SDS-PAGE gel of fractions froman anion exchange column as detailed in the examples. Lane 1: AAV vectorparticles; Lanes 2-5: vector elution fractions; Lane 6: proteinmolecular weight standards.

FIG. 4 is a depiction of a silver-stained SDS-PAGE gel showing elutionfractions (Lanes 12-21) from a cation exchange column as described inthe examples.

FIG. 5 is a depiction of a silver-stained SDS-PAGE gel showingseparation of AAV empty particles from AAV vector particles using cationexchange column chromatography. Lane 1: starting material; Lanes 2-4:three independent samples of vectors eluted from cation exchangecolumns.

DETAILED DESCRIPTION OF THE INVENTION

The practice of the present invention will employ, unless otherwiseindicated, conventional methods of virology, microbiology, molecularbiology and recombinant DNA techniques within the skill of the art. Suchtechniques are explained fully in the literature. See, e.g.; Sambrook etal. Molecular Cloning: A Laboratory Manual (Current Edition); DNACloning: A Practical Approach, Vol. I & II (D. Glover, ed.);Oligonucleotide Synthesis (N. Gait, ed., Current Edition); Nucleic AcidHybridization (B. Hames & S. Higgins, eds., Current Edition);Transcription and Translation (B. Hames & S. Higgins, eds., CurrentEdition); CRC Handbook of Parvoviruses, vol. I & II (P. Tijssen, ed.);Fundamental Virology, 2nd Edition, vol. I & II (B. N. Fields and D. M.Knipe, eds.); Freshney Culture of Animal Cells, A Manual of BasicTechnique (Wiley-Liss, Third Edition); and Ausubel et al. (1991) CurrentProtocols in Molecular Biology (Wiley Interscience, NY).

All publications, patents and patent applications cited herein, whethersupra or infra, are hereby incorporated by reference in their entirety.

1. Definitions

In describing the present invention, the following terms will beemployed, and are intended to be defined as indicated below.

It must be noted that, as used in this specification and the appendedclaims, the singular forms “a”, “an” and “the” include plural referentsunless the content clearly dictates otherwise. Thus, for example,reference to “a packaged capsid” includes a mixture of two or more suchcapsids, and the like.

By “vector” is meant any genetic element, such as a plasmid, phage,transposon, cosmid, chromosome, virus, virion, etc., which is capable ofreplication when associated with the proper control elements and whichcan transfer gene sequences between cells. Thus, the term includescloning and expression vehicles, as well as viral vectors.

By an “AAV vector” is meant a vector derived from an adeno-associatedvirus serotype, including without limitation, AAV-1, AAV-2, AAV-3,AAV-4, AAV-5, AAV-6, AAV-7 and AAV-8. AAV vectors can have one or moreof the AAV wild-type genes deleted in whole or part, preferably the repand/or cap genes, but retain functional flanking ITR sequences.Functional ITR sequences are necessary for the rescue, replication andpackaging of the AAV virion. Thus, an AAV vector is defined herein toinclude at least those sequences required in cis for replication andpackaging (e.g., functional ITRs) of the virus. The ITRs need not be thewild-type nucleotide sequences, and may be altered, e.g., by theinsertion, deletion or substitution of nucleotides, so long as thesequences provide for functional rescue, replication and packaging.

“AAV helper functions” refer to AAV-derived coding sequences which canbe expressed to provide AAV gene products that, in turn, function intrans for productive AAV replication. Thus, AAV helper functions includeboth of the major AAV open reading frames (ORFs), rep and cap. The Repexpression products have been shown to possess many functions,including, among others: recognition, binding and nicking of the AAVorigin of DNA replication; DNA helicase activity; and modulation oftranscription from AAV (or other heterologous) promoters. The Capexpression products supply necessary packaging functions. AAV helperfunctions are used herein to complement AAV functions in trans that aremissing from AAV vectors.

The term “AAV helper construct” refers generally to a nucleic acidmolecule that includes nucleotide sequences providing AAV functionsdeleted from an AAV vector which is to be used to produce a transducingvector for delivery of a nucleotide sequence of interest. AAV helperconstructs are commonly used to provide transient expression of AAV repand/or cap genes to complement missing AAV functions that are necessaryfor AAV replication; however, helper constructs lack AAV ITRs and canneither replicate nor package themselves. AAV helper constructs can bein the form of a plasmid, phage, transposon, cosmid, virus, or virion. Anumber of AAV helper constructs have been described, such as thecommonly used plasmids pAAV/Ad and pIM29+45 which encode both Rep andCap expression products. See, e.g., Samulski et al. (1989) J. Virol.63:3822-3828; and McCarty et al. (1991) J. Virol. 65:2936-2945. A numberof other vectors have been described which encode Rep and/or Capexpression products. See, e.g., U.S. Pat. Nos. 5,139,941 and 6,376,237.

The term “accessory functions” refers to non-AAV derived viral and/orcellular functions upon which AAV is dependent for its replication.Thus, the term captures proteins and RNAs that are required in AAVreplication, including those moieties involved in activation of AAV genetranscription, stage specific AAV mRNA splicing, AAV DNA replication,synthesis of Cap expression products and AAV capsid assembly.Viral-based accessory functions can be derived from any of the knownhelper viruses such as adenovirus, herpesvirus (other than herpessimplex virus type-1) and vaccinia virus.

The term “accessory function vector” refers generally to a nucleic acidmolecule that includes nucleotide sequences providing accessoryfunctions. An accessory function vector can be transfected into asuitable host cell, wherein the vector is then capable of supporting AAVvirion production in the host cell. Expressly excluded from the term areinfectious viral particles as they exist in nature, such as adenovirus,herpesvirus or vaccinia virus particles. Thus, accessory functionvectors can be in the form of a plasmid, phage, transposon or cosmid. Inparticular, it has been demonstrated that the full-complement ofadenovirus genes are not required for accessory helper functions. Forexample, adenovirus mutants incapable of DNA replication and late genesynthesis have been shown to be permissive for AAV replication. Ito etal., (1970) J. Gen. Virol. 9:243; Ishibashi et al, (1971) Virology45:317. Similarly, mutants within the E2B and E3 regions have been shownto support AAV replication, indicating that the E2B and E3 regions areprobably not involved in providing accessory functions. Carter et al.,(1983) Virology 126:505. However, adenoviruses defective in the E1region, or having a deleted E4 region, are unable to support AAVreplication. Thus, E1A and E4 regions are likely required for AAVreplication, either directly or indirectly. Laughlin et al., (1982) J.Virol. 41:868; Janik et al., (1981) Proc. Natl. Acad. Sci. USA 78:1925;Carter et al., (1983) Virology 126:505. Other characterized Ad mutantsinclude: E1B (Laughlin et al. (1982), supra; Janik et al. (1981), supra;Ostrove et al., (1980) Virology 104:502); E2A (Handa et al., (1975) J.Gen. Virol. 29:239; Strauss et al., (1976) J. Virol. 17:140; Myers etal., (1980) J. Virol. 35:665; Jay et al., (1981) Proc. Natl. Acad. Sci.USA 78:2927; Myers et al., (1981) J. Biol. Chem. 256:567); E2B (Carter,Adeno-Associated Virus Helper Functions, in I CRC Handbook ofParvoviruses (P. Tijssen ed., 1990)); E3 (Carter et al. (1983), supra);and E4 (Carter et al.(1983), supra; Carter (1995)). Although studies ofthe accessory functions provided by adenoviruses having mutations in theE1B coding region have produced conflicting results, Samulski et al.,(1988) J. Virol. 62:206-210, recently reported that E1B55k is requiredfor AAV virion production, while E1B19k is not. In addition,International Publication WO 97/17458 and Matshushita et al., (1998)Gene Therapy 5:938-945, describe accessory function vectors encodingvarious Ad genes. Particularly preferred accessory function vectorscomprise an adenovirus VA RNA coding region, an adenovirus E4 ORF6coding region, an adenovirus E2A 72 kD coding region, an adenovirus E1Acoding region, and an adenovirus E1B region lacking an intact E1B55kcoding region. Such vectors are described in International PublicationNo. WO 01/83797.

By “recombinant virus” is meant a virus that has been geneticallyaltered, e.g., by the addition or insertion of a heterologous nucleicacid construct into the particle.

By “AAV virion” is meant a complete virus particle, such as a wild-type(wt) AAV virus particle (comprising a linear, single-stranded AAVnucleic acid genome associated with an AAV capsid protein coat). In thisregard, single-stranded AAV nucleic acid molecules of eithercomplementary sense, e.g., “sense” or “antisense” strands, can bepackaged into any one AAV virion and both strands are equallyinfectious.

The terms “recombinant AAV virion,” “rAAV virion,” “AAV vectorparticle,” “full capsids,” “fulls,” and “full particles” are definedherein as an infectious, replication-defective virus including an AAVprotein shell, encapsidating a heterologous nucleotide sequence ofinterest which is flanked on both sides by AAV ITRs. A rAAV virion isproduced in a suitable host cell which has had sequences specifying anAAV vector, AAV helper functions and accessory functions introducedtherein. In this manner, the host cell is rendered capable of encodingAAV polypeptides that are required for packaging the AAV vector(containing a recombinant nucleotide sequence of interest) intoinfectious recombinant virion particles for subsequent gene delivery.

The terms “empty capsid,” “empty particle,” and “empties” refer to anAAV virion that includes an AAV protein shell but that lacks in whole orpart the polynucleotide construct comprising the heterologous nucleotidesequence of interest flanked on both sides by AAV ITRs. Accordingly, theempty capsid does not function to transfer the gene of interest into thehost cell.

The term “host cell” denotes, for example, microorganisms, yeast cells,insect cells, and mammalian cells, that can be, or have been, used asrecipients of an AAV helper construct, an AAV vector plasmid, anaccessory function vector, or other transfer DNA. The term includes theprogeny of the original cell which has been transfected. Thus, a “hostcell” as used herein generally refers to a cell which has beentransfected with an exogenous DNA sequence. It is understood that theprogeny of a single parental cell may not necessarily be completelyidentical in morphology or in genomic or total DNA complement as theoriginal parent, due to natural, accidental, or deliberate mutation.

The term “transfection” is used to refer to the uptake of foreign DNA bya cell, and a cell has been “transfected” when exogenous DNA has beenintroduced inside the cell membrane. A number of transfection techniquesare generally known in the art. See, e.g., Graham et al. (1973)Virology, 52 :456, Sambrook et al. (1989) Molecular Cloning, alaboratory manual, Cold Spring Harbor Laboratories, New York, Davis etal. (1986) Basic Methods in Molecular Biology, Elsevier, and Chu et al.(1981) Gene 13:197. Such techniques can be used to introduce one or moreexogenous DNA moieties into suitable host cells.

As used herein, the term “cell line” refers to a population of cellscapable of continuous or prolonged growth and division in vitro. Often,cell lines are clonal populations derived from a single progenitor cell.It is further known in the art that spontaneous or induced changes canoccur in karyotype during storage or transfer of such clonalpopulations. Therefore, cells derived from the cell line referred to maynot be precisely identical to the ancestral cells or cultures, and thecell line referred to includes such variants.

A stock or preparation of rAAV virions comprising AAV vector particles(packaged genomes) is “substantially free of” AAV empty capsids when atleast about 50%-99% or more of the virions present in the stock are rAAVvirions with packaged genomes (i.e., AAV vector particles). Preferably,the AAV vector particles comprise at least about 75% to 85%, morepreferably about 90% of the virions present in the stock, even morepreferably at least about 95%, or even 99% or more by weight of thevirions present in the stock, or any integer between these ranges. Thus,a stock is substantially free of AAV empty capsids when from about 40%to about 1% or less, preferably about 25% to about 15% or less, morepreferably about 10% or less, even more preferably about 5% to about 1%or less of the resulting stock comprises empty capsids.

A “nucleic acid” sequence refers to a DNA or RNA sequence. The termcaptures sequences that include any of the known base analogues of DNAand RNA such as, but not limited to 4-acetylcytosine,8-hydroxy-N6-methyladenosine, aziridinylcytosine, pseudoisocytosine,5-(carboxyhydroxylmethyl) uracil, 5-fluorouracil, 5-bromouracil,5-carboxymethylaminomethyl-2-thiouracil,5-carboxymethylaminomethyluracil, dihydrouracil, inosine,N6-isopentenyladenine, 1-methyladenine, 1-methylpseudouracil,1-methylguanine, 1-methylinosine, 2,2-dimethylguanine, 2-methyladenine,2-methylguanine, 3-methylcytosine, 5-methylcytosine, N6-methyladenine,7-methylguanine, 5-methylaminomethyluracil,5-methoxyaminomethyl-2-thiouracil, beta-D-mannosylqueosine,5′-methoxycarbonylmethyluracil, 5-methoxyuracil,2-methylthio-N6-isopentenyladenine, uracil-5-oxyacetic acid methylester,uracil-5-oxyacetic acid, oxybutoxosine, pseudouracil, queosine,2-thiocytosine, 5-methyl-2-thiouracil, 2-thiouracil, 4-thiouracil,5-methyluracil, Buracil-5-oxyacetic acid methylester, uracil-5-oxyaceticacid, pseudouracil, queosine, 2-thiocytosine, and 2,6-diaminopurine.

A “coding sequence” or a sequence which “encodes” a selectedpolypeptide, is a nucleic acid molecule which is transcribed (in thecase of DNA) and translated (in the case of mRNA) into a polypeptide invivo when placed under the control of appropriate regulatory sequences.The boundaries of the coding sequence are determined by a start codon atthe 5′ (amino) terminus and a translation stop codon at the 3′ (carboxy)terminus. A transcription termination sequence may be located 3′ to thecoding sequence.

The term DNA “control sequences” refers collectively to promotersequences, polyadenylation signals, transcription termination sequences,upstream regulatory domains, origins of replication, internal ribosomeentry sites (“IRES”), enhancers, and the like, which collectivelyprovide for the replication, transcription and translation of a codingsequence in a recipient cell. Not all of these control sequences needalways be present so long as the selected coding sequence is capable ofbeing replicated, transcribed and translated in an appropriate hostcell.

The term “promoter” is used herein in its ordinary sense to refer to anucleotide region comprising a DNA regulatory sequence, wherein theregulatory sequence is derived from a gene which is capable of bindingRNA polymerase and initiating transcription of a downstream(3′-direction) coding sequence. Transcription promoters can include“inducible promoters” (where expression of a polynucleotide sequenceoperably linked to the promoter is induced by an analyte, cofactor,regulatory protein, etc.), “repressible promoters” (where expression ofa polynucleotide sequence operably linked to the promoter is induced byan analyte, cofactor, regulatory protein, etc.), and “constitutivepromoters.”

“Operably linked” refers to an arrangement of elements wherein thecomponents so described are configured so as to perform their usualfunction. Thus, control sequences operably linked to a coding sequenceare capable of effecting the expression of the coding sequence. Thecontrol sequences need not be contiguous with the coding sequence, solong as they function to direct the expression thereof. Thus, forexample, intervening untranslated yet transcribed sequences can bepresent between a promoter sequence and the coding sequence and thepromoter sequence can still be considered “operably linked” to thecoding sequence.

For the purpose of describing the relative position of nucleotidesequences in a particular nucleic acid molecule throughout the instantapplication, such as when a particular nucleotide sequence is describedas being situated “upstream,” “downstream,” “3′,” or “5′” relative toanother sequence, it is to be understood that it is the position of thesequences in the “sense” or “coding” strand of a DNA molecule that isbeing referred to as is conventional in the art.

The term “heterologous” as it relates to nucleic acid sequences such ascoding sequences and control sequences, denotes sequences that are notnormally joined together, and/or are not normally associated with aparticular cell. Thus, a “heterologous” region of a nucleic acidconstruct or a vector is a segment of nucleic acid within or attached toanother nucleic acid molecule that is not found in association with theother molecule in nature. For example, a heterologous region of anucleic acid construct could include a coding sequence flanked bysequences not found in association with the coding sequence in nature.Another example of a heterologous coding sequence is a construct wherethe coding sequence itself is not found in nature (e.g., syntheticsequences having codons different from the native gene). Similarly, acell transformed with a construct which is not normally present in thecell would be considered heterologous for purposes of this invention.Allelic variation or naturally occurring mutational events do not giverise to heterologous DNA, as used herein.

By “isolated” when referring to a nucleotide sequence, is meant that theindicated molecule is present in the substantial absence of otherbiological macromolecules of the same type. Thus, an “isolated nucleicacid molecule which encodes a particular polypeptide” refers to anucleic acid molecule which is substantially free of other nucleic acidmolecules that do not encode the subject polypeptide; however, themolecule may include some additional bases or moieties which do notdeleteriously affect the basic characteristics of the composition.

2. Modes of Carrying Out the Invention

Before describing the present invention in detail, it is to beunderstood that this invention is not limited to particular formulationsor process parameters as such formulations or process parameters may, ofcourse, vary. It is also to be understood that the terminology usedherein is for the purpose of describing particular embodiments of theinvention only, and is not intended to be limiting.

Although a number of methods and materials similar or equivalent tothose described herein can be used in the practice of the presentinvention, the preferred materials and methods are described herein.

The present invention involves reducing the numbers of, or eliminating,AAV empty capsids contained within purified stocks of AAV virions, withminimal loss to AAV vector particles contained therein. The methods ofthe present invention may be used regardless of the process in whichrAAV virions are generated.

There are several methods that are well known in the art for generatingrAAV virions: for example, transfection using vector and AAV helpersequences in conjunction with coinfection with one of the AAV helperviruses (e.g., adenovirus, herpesvirus, or vaccinia virus) ortransfection with a recombinant AAV vector, an AAV helper vector, and anaccessory function vector. For detailed descriptions of methods forgenerating rAAV virions see, U.S. Pat. Nos. 6,001,650 and 6,004,797,both incorporated herein by reference in their entireties.

For example, wild-type AAV and helper viruses may be used to provide thenecessary replicative functions for producing rAAV virions (see, e.g.,U.S. Pat. No. 5,139,941, incorporated herein by reference in itsentirety). Alternatively, a plasmid, containing helper function genes,in combination with infection by one of the well-known helper virusescan be used as the source of replicative functions (see e.g., U.S. Pat.Nos. 5,622,856 and 5,139,941, both incorporated herein by reference intheir entireties). Similarly, a plasmid, containing accessory functiongenes can be used in combination with infection by wild-type AAV, toprovide the necessary replicative functions. These three approaches,when used in combination with a rAAV vector, are each sufficient toproduce rAAV virions. Other approaches, well known in the art, can alsobe employed by the skilled artisan to produce rAAV virions.

In a preferred embodiment of the present invention, a tripletransfection method (described in detail in U.S. Pat. No. 6,001,650,incorporated by reference herein in its entirety) is used to producerAAV virions because this method does not require the use of aninfectious helper virus, enabling rAAV virions to be produced withoutany detectable helper virus present. This is accomplished by use ofthree vectors for rAAV virion production: an AAV helper function vector,an accessory function vector, and a rAAV expression vector. One of skillin the art will appreciate, however, that the nucleic acid sequencesencoded by these vectors can be provided on two or more vectors invarious combinations.

As explained herein, the AAV helper function vector encodes the “AAVhelper function” sequences (i.e., rep and cap), which function in transfor productive AAV replication and encapsidation. Preferably, the AAVhelper function vector supports efficient AAV vector production withoutgenerating any detectable wt AAV virions (i.e., AAV virions containingfunctional rep and cap genes). An example of such a vector, pHLP19 isdescribed in U.S. Pat. No. 6,001,650, incorporated herein by referencein its entirety. The rep and cap genes of the AAV helper function vectorcan be derived from any of the known AAV serotypes, as explained above.For example, the AAV helper function vector may have a rep gene derivedfrom AAV-2 and a cap gene derived from AAV-6; one of skill in the artwill recognize that other rep and cap gene combinations are possible,the defining feature being the ability to support rAAV virionproduction.

The accessory function vector encodes nucleotide sequences for non-AAVderived viral and/or cellular functions upon which AAV is dependent forreplication (i.e., “accessory functions”). The accessory functionsinclude those functions required for AAV replication, including, withoutlimitation, those moieties involved in activation of AAV genetranscription, stage specific AAV mRNA splicing, AAV DNA replication,synthesis of cap expression products, and AAV capsid assembly.Viral-based accessory functions can be derived from any of thewell-known helper viruses such as adenovirus, herpesvirus (other thanherpes simplex virus type-1), and vaccinia virus. In a preferredembodiment, the accessory function plasmid pLadeno5 is used (detailsregarding pLadeno5 are described in U.S. Pat. No. 6,004,797,incorporated herein by reference in its entirety). This plasmid providesa complete set of adenovirus accessory functions for AAV vectorproduction, but lacks the components necessary to formreplication-competent adenovirus.

Once stocks of AAV virions are produced, a number of methods, detailedbelow, can be used to determine infectious titers and to purify AAVvector particles away from AAV empty capsids.

In order to further an understanding of the invention, a more detaileddiscussion is provided below regarding recombinant AAV expressionvectors, AAV helper and accessory functions, compositions comprising AAVvirions, as well as delivery of virions.

Recombinant AAV Expression Vectors

Recombinant AAV (rAAV) expression vectors are constructed using knowntechniques to provide as operatively linked components in the directionof transcription, control elements including a transcriptionalinitiation region, the polynucleotide of interest and a transcriptionaltermination region. The control elements are selected to be functionalin the host cell of interest. The resulting construct which contains theoperatively linked components is bounded (5′ and 3′) with functional AAVITR sequences.

The nucleotide sequences of AAV ITR regions are known. See, e.g., Kotin,R. M. (1994) Human Gene Therapy 5:793-801; Berns, K. I. “Parvoviridaeand their Replication” in Fundamental Virology, 2nd Edition, (B. N.Fields and D. M. Knipe, eds.) for the AAV-2 sequence. AAV ITRs used inthe vectors of the invention need not have a wild-type nucleotidesequence, and may be altered, e.g., by the insertion, deletion orsubstitution of nucleotides. Additionally, AAV ITRs may be derived fromany of several AAV serotypes, including without limitation, AAV-1,AAV-2, AAV-3, AAV-4, AAV-5, AAV-6, AAV-7 and AAV-8, etc. Furthermore, 5′and 3′ ITRs which flank a selected nucleotide sequence in an AAVexpression vector need not necessarily be identical or derived from thesame AAV serotype or isolate, so long as they function as intended,i.e., to allow for excision and rescue of the sequence of interest froma host cell genome or vector, and to allow integration of the DNAmolecule into the recipient cell genome when AAV Rep gene products arepresent in the cell.

Suitable polynucleotide molecules for use in AAV vectors will be lessthan about 5 kilobases (kb) in size. The selected polynucleotidesequence is operably linked to control elements that direct thetranscription or expression thereof in the subject in vivo. Such controlelements can comprise control sequences normally associated with theselected gene. Alternatively, heterologous control sequences can beemployed. Useful heterologous control sequences generally include thosederived from sequences encoding mammalian or viral genes. Examplesinclude, but are not limited to, neuron-specific enolase promoter, aGFAP promoter, the SV40 early promoter, mouse mammary tumor virus LTRpromoter; adenovirus major late promoter (Ad MLP); a herpes simplexvirus (HSV) promoter, a cytomegalovirus (CMV) promoter such as the CMVimmediate early promoter region (CMVIE), a rous sarcoma virus (RSV)promoter, the CAG promoter, synthetic promoters, hybrid promoters, andthe like. In addition, sequences derived from nonviral genes, such asthe murine metallothionein gene, will also find use herein. Suchpromoter sequences are commercially available from, e.g., Stratagene(San Diego, Calif.).

The AAV expression vector which harbors the polynucleotide molecule ofinterest bounded by AAV ITRs, can be constructed by directly insertingthe selected sequence(s) into an AAV genome which has had the major AAVopen reading frames (“ORFs”) excised therefrom. Other portions of theAAV genome can also be deleted, so long as a sufficient portion of theITRs remain to allow for replication and packaging functions. Suchconstructs can be designed using techniques well known in the art. See,e.g., U.S. Pat. Nos. 5,173,414 and 5,139,941; International PublicationNos. WO 92/01070 (published 23 Jan. 1992) and WO 93/03769 (published 4Mar. 1993); Lebkowski et al. (1988) Molec. Cell. Biol. 8:3988-3996;Vincent et al. (1990) Vaccines 90 (Cold Spring Harbor Laboratory Press);Carter (1992) Current Opinion in Biotechnology 3:533-539; Muzyczka(1992) Current Topics in Microbiol. and Immunol. 158:97-129; Kotin(1994) Human Gene Therapy 5:793-801; Shelling and Smith (1994) GeneTherapy 1:165-169; and Zhou et al. (1994) J. Exp. Med. 179:1867-1875.

Alternatively, AAV ITRs can be excised from the viral genome or from anAAV vector containing the same and fused 5′ and 3′ of a selected nucleicacid construct that is present in another vector using standard ligationtechniques, such as those described in Sambrook et al., supra. Forexample, ligations can be accomplished in 20 mM Tris-Cl pH 7.5, 10 mMMgCl2, 10 mM DTT, 33 μg/ml BSA, 10 mM-50 mM NaCl, and either 40 μM ATP,0.01-0.02 (Weiss) units T4 DNA ligase at 0° C. (for “sticky end”ligation) or 1 mM ATP, 0.3-0.6 (Weiss) units T4 DNA ligase at 14° C.(for “blunt end” ligation). Intermolecular “sticky end” ligations areusually performed at 30-100 μg/ml total DNA concentrations (5-100 nMtotal end concentration). AAV vectors which contain ITRs have beendescribed in, e.g., U.S. Pat. No. 5,139,941. In particular, several AAVvectors are described therein which are available from the American TypeCulture Collection (“ATCC”) under Accession Numbers 53222, 53223, 53224,53225 and 53226.

For the purposes of the invention, suitable host cells for producingrAAV virions from the AAV expression vectors include microorganisms,yeast cells, insect cells, and mammalian cells, that can be, or havebeen, used as recipients of a heterologous DNA molecule and that arecapable of growth in, for example, suspension culture, a bioreactor, orthe like. The term includes the progeny of the original cell which hasbeen transfected. Thus, a “host cell” as used herein generally refers toa cell which has been transfected with an exogenous DNA sequence. Cellsfrom the stable human cell line, 293 (readily available through, e.g.,the American Type Culture Collection under Accession Number ATCCCRL1573) are preferred in the practice of the present invention.Particularly, the human cell line 293 is a human embryonic kidney cellline that has been transformed with adenovirus type-5 DNA fragments(Graham et al. (1977) J. Gen. Virol. 36:59), and expresses theadenoviral E1a and E1b genes (Aiello et al. (1979) Virology 94:460). The293 cell line is readily transfected, and provides a particularlyconvenient platform in which to produce rAAV virions.

AAV Helper Functions

Host cells containing the above-described AAV expression vectors must berendered capable of providing AAV helper functions in order to replicateand encapsidate the nucleotide sequences flanked by the AAV ITRs toproduce rAAV virions. AAV helper functions are generally AAV-derivedcoding sequences which can be expressed to provide AAV gene productsthat, in turn, function in trans for productive AAV replication. AAVhelper functions are used herein to complement necessary AAV functionsthat are missing from the AAV expression vectors. Thus, AAV helperfunctions include one, or both of the major AAV ORFs, namely the rep andcap coding regions, or functional homologues thereof.

By “AAV rep coding region” is meant the art-recognized region of the AAVgenome which encodes the replication proteins Rep 78, Rep 68, Rep 52 andRep 40. These Rep expression products have been shown to possess manyfunctions, including recognition, binding and nicking of the AAV originof DNA replication, DNA helicase activity and modulation oftranscription from AAV (or other heterologous) promoters. The Repexpression products are collectively required for replicating the AAVgenome. For a description of the AAV rep coding region, see, e.g.,Muzyczka, N. (1992) Current Topics in Microbiol. and Immunol.158:97-129; and Kotin, R. M. (1994) Human Gene Therapy 5:793-801.Suitable homologues of the AAV rep coding region include the humanherpesvirus 6 (HHV-6) rep gene which is also known to mediate AAV-2 DNAreplication (Thomson et al. (1994) Virology 204:304-311).

By “AAV cap coding region” is meant the art-recognized region of the AAVgenome which encodes the capsid proteins VP1, VP2, and VP3, orfunctional homologues thereof. These Cap expression products supply thepackaging functions which are collectively required for packaging theviral genome. For a description of the AAV cap coding region, see, e.g.,Muzyczka, N. and Kotin, R. M. (supra).

AAV helper functions are introduced into the host cell by transfectingthe host cell with an AAV helper construct either prior to, orconcurrently with, the transfection of the AAV expression vector. AAVhelper constructs are thus used to provide at least transient expressionof AAV rep and/or cap genes to complement missing AAV functions that arenecessary for productive AAV infection. AAV helper constructs lack AAVITRs and can neither replicate nor package themselves.

These constructs can be in the form of a plasmid, phage, transposon,cosmid, virus, or virion. A number of AAV helper constructs have beendescribed, such as the commonly used plasmids pAAV/Ad and pIM29+45 whichencode both Rep and Cap expression products. See, e.g., Samulski et al.(1989) J. Virol. 63:3822-3828; and McCarty et al. (1991) J. Virol.65:2936-2945. A number of other vectors have been described which encodeRep and/or Cap expression products. See, e.g., U.S. Pat. No. 5,139,941.

AAV Accessory Functions

The host cell (or packaging cell) must also be rendered capable ofproviding nonAAV-derived functions, or “accessory functions,” in orderto produce rAAV virions. Accessory functions are nonAAV-derived viraland/or cellular functions upon which AAV is dependent for itsreplication. Thus, accessory functions include at least those nonAAVproteins and RNAs that are required in AAV replication, including thoseinvolved in activation of AAV gene transcription, stage specific AAVmRNA splicing, AAV DNA replication, synthesis of Cap expression productsand AAV capsid assembly. Viral-based accessory functions can be derivedfrom any of the known helper viruses.

In particular, accessory functions can be introduced into and thenexpressed in host cells using methods known to those of skill in theart. Typically, accessory functions are provided by infection of thehost cells with an unrelated helper virus. A number of suitable helperviruses are known, including adenoviruses; herpesviruses such as herpessimplex virus types 1 and 2; and vaccinia viruses. Nonviral accessoryfunctions will also find use herein, such as those provided by cellsynchronization using any of various known agents. See, e.g., Buller etal. (1981) J. Virol. 40:241-247; McPherson et al. (1985) Virology147:217-222; Schlehofer et al. (1986) Virology 152:110-117.

Alternatively, accessory functions can be provided using an accessoryfunction vector as defined above. See, e.g., U.S. Pat. No. 6,004,797 andInternational Publication No. WO 01/83797, incorporated herein byreference in its entirety. Nucleic acid sequences providing theaccessory functions can be obtained from natural sources, such as fromthe genome of an adenovirus particle, or constructed using recombinantor synthetic methods known in the art. As explained above, it has beendemonstrated that the full-complement of adenovirus genes are notrequired for accessory helper functions. In particular, adenovirusmutants incapable of DNA replication and late gene synthesis have beenshown to be permissive for AAV replication. Ito et al., (1970) J. Gen.Virol. 9:243; Ishibashi et al, (1971) Virology 45:317. Similarly,mutants within the E2B and E3 regions have been shown to support AAVreplication, indicating that the E2B and E3 regions are probably notinvolved in providing accessory functions. Carter et al., (1983)Virology 126:505. However, adenoviruses defective in the E1 region, orhaving a deleted E4 region, are unable to support AAV replication. Thus,E1A and E4 regions are likely required for AAV replication, eitherdirectly or indirectly. Laughlin et al., (1982) J. Virol. 41:868; Janiket al., (1981) Proc. Natl. Acad. Sci. USA 78:1925; Carter et al., (1983)Virology 126:505. Other characterized Ad mutants include: E1B (Laughlinet al. (1982), supra; Janik et al. (1981), supra; Ostrove et al., (1980)Virology 104:502); E2A (Handa et al., (1975) J. Gen. Virol. 29:239;Strauss et al., (1976) J. Virol. 17:140; Myers et al., (1980) J. Virol.35:665; Jay et al., (1981) Proc. Natl. Acad. Sci. USA 78:2927; Myers etal., (1981) J. Biol. Chem. 256:567); E2B (Carter, Adeno-Associated VirusHelper Functions, in I CRC Handbook of Parvoviruses (P. Tijssen ed.,1990)); E3 (Carter et al. (1983), supra); and E4 (Carter et al. (1983),supra; Carter (1995)). Although studies of the accessory functionsprovided by adenoviruses having mutations in the E1B coding region haveproduced conflicting results, Samulski et al., (1988) J. Virol.62:206-210, recently reported that E1B55k is required for AAV virionproduction, while E1B19k is not. In addition, International PublicationWO 97/17458 and Matshushita et al., (1998) Gene Therapy 5:938-945,describe accessory function vectors encoding various Ad genes.Particularly preferred accessory function vectors comprise an adenovirusVA RNA coding region, an adenovirus E4 ORF6 coding region, an adenovirusE2A 72 kD coding region, an adenovirus E1A coding region, and anadenovirus E1B region lacking an intact E1B55k coding region. Suchvectors are described in International Publication No. WO 01/83797.

As a consequence of the infection of the host cell with a helper virus,or transfection of the host cell with an accessory function vector,accessory functions are expressed which transactivate the AAV helperconstruct to produce AAV Rep and/or Cap proteins. The Rep expressionproducts excise the recombinant DNA (including the DNA of interest) fromthe AAV expression vector. The Rep proteins also serve to duplicate theAAV genome. The expressed Cap proteins assemble into capsids, and therecombinant AAV genome is packaged into the capsids. Thus, productiveAAV replication ensues, and the DNA is packaged into rAAV virions.

Purification of rAAV Virions

Following recombinant AAV replication, rAAV virions can be purified fromthe host cell using a variety of conventional purification methods, suchas column chromatography, CsCl gradients, and the like. For example, aplurality of column purification steps can be used, such as purificationover an anion exchange column, an affinity column and/or a cationexchange column. See, for example, International Publication No. WO02/12455. Further, if infection is employed to express the accessoryfunctions, residual helper virus can be inactivated, using knownmethods. For example, adenovirus can be inactivated by heating totemperatures of approximately 60° C. for, e.g., 20 minutes or more. Thistreatment effectively inactivates only the helper virus since AAV isextremely heat stable while the helper adenovirus is heat labile.

Recombinant AAV vectors containing any number of reporter genes can beused to determine infectious titers. For example, alkaline phosphatase,β-galactosidase (LacZ), green fluorescent protein, or luciferase may beused. After harvesting the transfected host cell, a lysate is formed bydisrupting the transfected host cells using techniques suitable forlarge-scale production, such as microfluidization. The lysate is thenfiltered (for example, through a 0.45 μm filter), and purified usingcolumn chromatographic methods as described herein. Other techniqueshave also been reported to determine the infectious titer of any AAVvector. See, e.g., Zhen et al., “An Infectious Titer Assay forAdeno-associated Virus (AAV) Vectors with Sensitivity Sufficient toDetect Single Infectious Events.” Hum. Gene Ther. (2004) In Press.

The purified AAV stock is then treated to remove empty capsids usingcolumn chromatography techniques. In a particularly preferred method ofthe invention, rAAV preparations are obtained by lysing transfectedcells to obtain a crude cell lysate. The crude cell lysate can then beclarified to remove cell debris by techniques well known in the art,such as filtering, centrifuging, and the like, to render a clarifiedcell lysate. The crude cell lysate or clarified cell lysate, whichcontain both AAV vector particles and AAV empty capsids, is then appliedto a first cation exchange column under non-separating conditions. Thefirst cation exchange column functions to further separate the AAVvector particles and the AAV empty capsids from cellular and othercomponents present in the cell lysate preparation. Methods forperforming the initial purification of the cell lysate are known. Onerepresentative method is described in U.S. Pat. No. 6,593,123,incorporated herein by reference in its entirety.

Fractions collected from the first cation exchange column are thenapplied to a second ion exchanger, i.e., a second cation exchange columnand/or an anion exchange column, using different elution conditions thatseparate AAV empty capsids from AAV vector particles.

Suitable cation exchangers for both the first cation exchange column andthe second cation exchange column, if used, include a wide variety ofmaterials, known in the art. Particularly preferred are strong cationexchangers capable of binding rAAV virions over a wide pH range. Forexample carboxymethylated and sulfonated cation exchange matrices areparticularly useful for use herein. Useful matrix materials include butare not limited to, cellulose matrices, such as fibrous, microgranularand beaded matrices; agarose, dextran, polyacrylate, polyvinyl,polystyrene, silica and polyether matrices; and composites. Particularlypreferred herein are matrices containing the functional ligand R—SO₃ ⁻,preferably sulfopropyl or sulfoethyl resins. Representative matricesinclude but are not limited to POROS HS, POROS SP, POROS S (all strongcation exchangers available from Applied Biosystems, Foster City,Calif.), POROS CM (weak cation exchanger available from AppliedBiosystems, Foster City, Calif.), TOSOHAAS TOYOPEARL SP550C and MERCKFRACTOGEL EMD SO₃ ⁻-650(m), as well as SOURCE 15S, SOURCE 30S, SEPHAROSESP FF, SEPHAROSE SP XL (all available from Amersham Bioscience,Piscataway, N.J.).

For all column chromatography protocols given below, columns can beprepared using standard protocols known in the art with the appropriatebuffer solutions. Sample is then loaded. For the first cation exchangecolumn used, conditions are such that both empty capsids and AAV vectorparticles bind to the column resin and are subsequently eluted together,but are separated from other cellular components and debris present inthe cell lysate. For example, empty and full capsids are eluted using abuffer of appropriate ionic strength. Suitable buffers include e.g.,10-50 mM sodium phosphate, preferably 15-40, such as 15 . . . 20 . . .25 . . . 30 . . . 35 . . . 40, etc. mM sodium phosphate containing salt,such as NaCl or KCl, at a concentration of e.g, 100-700 mM, such as200-400 mM, e.g., 200 . . . 300 . . . 325 . . . 350 . . . 370 . . . 380. . . 400, etc., or any concentration within these ranges. The pH of thebuffer can be from about 3 to about 9.5, such as 4-8, e.g., pH 4 . . .4.5 . . . 5 . . . 5.5 . . . 6, etc., or any pH within these ranges. Thefractions are collected and then can be run either on an anion exchangecolumn and/or a second cation exchange column under separatingconditions.

If a second cation exchange column is used in a subsequent step toseparate empty AAV capsids from AAV vector particles, two elutionbuffers are used, one low salt buffer and one high salt buffer. Inparticular, empty capsids are separated from AAV vector particles usingan appropriate buffer at a pH of from about pH 6 to pH 12, preferably pH7 to pH 10, and even more preferably pH 7.5 to pH 9.5, such as pH 7.5 .. . 8.0, 8.1, 8.2, 8.3, 8.4, 8.5 . . . 9.0, 9.1, 9.2, 9.3, 9.4, 9.5, orany pH between the stated ranges. Appropriate buffers are well known inthe art and include, without limitation, buffers with the followingbuffer ions: acetic acid; malonic acid; MES; phosphate; HEPES, BICINE,and the like. To elute the sample, the ionic strength of the startingbuffer is increased using a salt, such as NaCl, KCl, ammonia sulfate orany other salts containing sulfate, formate, acetate, citrate, and/orphosphate. In one embodiment of the invention, the column is firsttreated with a low salt concentration, e.g., 10-200 mM of ammoniumacetate, such as 20 . . . 25 . . . 30 . . . 35 . . . 40 . . . 45 . . .50 . . . 55 . . . 60 . . . 65 . . . 100 mM, or any concentration withinthese ranges. This treatment results in elution of AAV vector particlesfrom the column resin. Subsequently the column is treated with a highersalt concentration in order to elute AAV empty capsids. One example foruse as the second buffer is ammonium acetate with a concentration of100-800 mM, preferably 500-700 mM, such as 500 . . . 550 . . . 600 . . .650 . . . 700 . . . 800 mM, or any concentration within these statedranges. Using these conditions, the AAV vector particles elute in theearly fractions and the empty particles later.

As explained above, in an alternate method of the invention, thepreparation from the first cation exchange column is applied to an anionexchange column either in place of or in addition to the second cationexchange column. If an anion exchange column is used in addition to thesecond cation exchange column, it can be used either prior or subsequentto the second cation exchange column. Moreover, a second anion exchangecolumn can be used after the first anion exchange column. A number ofsuitable anion exchangers for use with the present invention are knownand include without limitation, MACRO PREP Q (strong anion-exchangeravailable from BioRad, Hercules, Calif.); UNOSPHERE Q (stronganion-exchanger available from BioRad, Hercules, Calif.); POROS 50HQ(strong anion-exchanger available from Applied Biosystems, Foster City,Calif.); POROS 50D (weak anion-exchanger available from AppliedBiosystems, Foster City, Calif.); POROS 50PI (weak anion-exchangeravailable from Applied Biosystems, Foster City, Calif.); SOURCE 30Q(strong anion-exchanger available from Amersham Biosciences, Piscataway,N.J.); DEAE SEPHAROSE (weak anion-exchanger available from AmershamBiosciences, Piscataway, N.J.); Q SEPHAROSE (strong anion-exchangeravailable from Amersham Biosciences, Piscataway, N.J.).

The anion exchange column is first equilibrated using standard buffersand according to the manufacturer's specifications. For example, thecolumn can be equilibrated with, e.g., a 5 to 50 mM, preferably 7-20 mM,such as 10 mM, sodium phosphate buffer. Sample is then loaded and twoelution buffers are used, one low salt buffer and one high salt buffer.Fractions are collected following each of the low salt and high saltwashes and protein is detected in the fractions using standardtechniques, such as monitoring UV absorption at 260 and 280 nm. Using ananion exchanger, the protein peaks from the lower salt eluate containAAV empty capsids and the higher salt fractions contain AAV vectorparticles.

In particular, on the anion exchange column, empty capsids can separatedfrom AAV vector particles using an appropriate buffer at a pH of fromabout pH 5 to pH 12, preferably pH 6 to pH 10, and even more preferablypH 7 to pH 9.5, such as pH 7.1, 7.2, 7.3, 7.4 . . . 8.0, 8.1, 8.2, 8.3,8.4, 8.5 . . . 9.0, 9.1, 9.2, 9.3, 9.4, 9.5, or any pH between thestated ranges. Appropriate buffers for use with the anion exchangecolumns are well known in the art and are generally cationic orzwitterionic in nature. Such buffers include, without limitation,buffers with the following buffer ions: N-methylpiperazine; piperazine;Bis-Tris; Bis-Tris propane; Triethanolamine; Tris;N-methyldiethanolamine; 1,3-diaminopropane; ethanolamine; acetic acid,and the like. To elute the sample, the ionic strength of the startingbuffer is increased using a salt, such as NaCl, KCl, sulfate, formate oracetate, at an appropriate pH.

In one embodiment of the invention, the anion exchange column is firsttreated with a low salt concentration, e.g., 10-100 mM of NaCl, such as10 . . . 20 . . . 25 . . . 30 . . . 35 . . . 40 . . . 45 . . . 50 . . .55 . . . 60 . . . 65 . . . 100 mM, or any concentration within theseranges. Following initial treatment, the column is then treated with ahigher salt concentration in order to elute empty capsids, such as ahigher NaCl concentration, or with another buffer with a greater ionicstrength. One example for use as the second buffer is a sodium acetatebuffer or a Tris-based buffer with a concentration of 100-300 mM,preferably 125-200 mM, such as 125 . . . 130 . . . 140 . . . 150 . . .160 . . . 170 . . . 180 . . . 190 . . . 200 mM, or any concentrationwithin these stated ranges. After the empty capsids are eluted from thecolumn, the AAV vector particles can be recovered using a higherconcentration of salt. One example for use as the elution buffer is 10mM Tris buffer containing sodium acetate at a concentration in the rangeof 100-500 mM, preferably 130-300 mM, such as 100 . . . 130 . . . 150 .. . 200 . . . 250 . . . 300 . . . 350 . . . 400 . . . 450 . . . 500 mM,or any concentration within these stated ranges.

Using the techniques described above, more than 90% of the AAV emptycapsids can be separated away from the AAV vector particles. Moreover,high recovery of AAV vector particles is readily achieved, i.e., morethan 10%, preferably more than 25%, even more preferably more than 50%,such as more than 60% of the AAV vector particles can be recovered.

Methods for assaying for empty capsids and AAV vector particles withpackaged genomes are known in the art. See, e.g., Grimm et al., GeneTherapy (1999) 6:1322-1330; Sommer et al., Molec. Ther. (2003)7:122-128. To test for denatured capsid, the methods include subjectingthe treated AAV stock to SDS-polyacrylamide gel electrophoresis,consisting of any gel capable of separating the three capsid proteins,for example, a gradient gel containing 3-8% Tris-acetate in the buffer,then running the gel until sample material is separated, and blottingthe gel onto nylon or nitrocellulose membranes, preferably nylon.Anti-AAV capsid antibodies are then used as the primary antibodies thatbind to denatured capsid proteins, preferably an anti-AAV capsidmonoclonal antibody, most preferably the B1 anti-AAV-2 monoclonalantibody (Wobus et al., J. Virol. (2000) 74:9281-9293). A secondaryantibody is then used, one that binds to the primary antibody andcontains a means for detecting binding with the primary antibody, morepreferably an anti-IgG antibody containing a detection moleculecovalently bound to it, most preferably a sheep anti-mouse IgG antibodycovalently linked to horseradish peroxidase. A method for detectingbinding is used to semi-quantitatively determine binding between theprimary and secondary antibodies, preferably a detection method capableof detecting radioactive isotope emissions, electromagnetic radiation,or colorimetric changes, most preferably a chemiluminescence detectionkit.

To test for infectious titer, the methods include the seeding of about100,000 host cells, preferably of human origin, most preferably HeLacells, into tissue culture-treated plates, preferably 24-well tissueculture-treated plates, and incubated for about 24 hours after whichadenovirus, preferably the adenovirus-2 serotype, and treated rAAV stockis added to the host cells. The host cells, adenovirus, and rAAV stockare allowed to incubate for 24 hours, after which the host cells arefixed, preferably with formaldehyde and glutaraldehyde, and stained withan appropriate agent that will detect the rAAV expressed transgene; forexample, with rAAV-LacZ, X-gal is contemplated as the staining agent.Other agents for other reporter genes are well known in the art. Moregeneral methods to determine infectivity titers of vectors containingany transgene are also known in the art. See, e.g., Zhen et al., “AnInfectious Titer Assay for Adeno-associated Virus (AAV) Vectors withSensitivity Sufficient to Detect Single Infectious Events.” Hum. GeneTher. (2004) In Press.

3. Experimental

Below are examples of specific embodiments for carrying out the presentinvention. The examples are offered for illustrative purposes only, andare not intended to limit the scope of the present invention in any way.

Efforts have been made to ensure accuracy with respect to numbers used(e.g., amounts, temperatures, etc.), but some experimental error anddeviation should, of course, be allowed for.

Material and Methods:

rAAV Production and Purification:

Recombinant AAV virions containing the gene encoding human factor IX(rAAV-hFIX) were produced by the triple-transfection method described inU.S. Pat. Nos. 6,001,650 and 6,004,797, both incorporated herein byreference in their entireties. The plasmids used were the accessoryfunction plasmid ‘pladeno5’, the AAV helper function plasmid ‘pHLP19’,and the recombinant AAV plasmid ‘phFIX-16’. Human embryonic kidney (HEK)293 cells were used as host cells for the production of rAAV virions.

The transfected 293 cells were harvested approximately 72 hourspost-transfection and disrupted by microfluidization using aMicrofluidizer™ (Microfluidics International Corp., Newton, Mass.) andthe crude lysate was collected and filtered through serial filters toremove cell debris to create a clarified cell lysate. The clarified celllysate containing packaged and empty capsids was then loaded on a POROS50HS column to purify AAV vector particles and AAV empty capsids fromother cellular components. The AAV vector particles and empty capsidswere eluted from the POROS 50HS column using a buffer containing 20 mMsodium phosphate, 370 mM NaCl, pH 5.5. The AAV preparation containingboth AAV vector particles and empty capsids was diluted 2-fold to reducethe salt concentration and further purified through a Q SEPHAROSEcolumn, the flow-through AAV vector particles and empty capsids werecollected, and concentration and buffer exchange was performed usingultra-filtration and dialfiltration techniques.

The final POROS 50HS column-purified product contained both AAV emptycapsids as well as AAV vector particles containing recombinant vectorgenomes. In order to generate control samples for each, cesium chloridewas added to the POROS 50HS column-purified material to a density of1.41 g/ml with mixing, and then subjected to ultracentrifugation at45,000 rpm using a Ti 70 rotor for 23 hours. The visible empty and AAVvector particle bands were drawn using syringes separately. The purifiedempty capsids and AAV vector particles were separately dialyzed againstbuffer composed of 10 mM sodium phosphate pH 7.4, 140 mM NaCl and 5%sorbital using dialysis cassettes (Pierce) at 4° C. with three bufferexchanges.

Resins and Column Chromatography:

Various types of chromatography resins including anion exchange, cationexchange resins, a chromatofocusing resin and hydrophobic interaction(HIC) resins, were tested for their potential to separate AAV emptycapsids and AAV vector particles. A high-throughput manifold was usedfor resin screening. Resins were packed into a disposable empty columnwith bed height of 3 cm (Bio-Rad Laboratories) that were installed on amanifold. The columns were equilibrated using buffer containing 10 mMsodium phosphate. The 0.2 μm filtered clarified HEK293 cell lysate AAVvectors and empty capsids were then diluted in column equilibrationbuffer a NaCl (salt) concentration of 50 mM and then loaded on thecolumns. The columns were washed with the equilibration buffer first andfurther washed with buffer containing 10 mM sodium phosphate and 40 mMNaCl pH 7.4 (low salt wash) to wash away unbound materials. Columns werethen treated with elution buffer containing 10 mM sodium phosphate and 1M NaCl (high salt wash). The materials washed off using the low saltwash were collected as one fraction, and the materials eluted using highsalt wash as a separate fraction.

Following the initial screening using disposable columns, larger scalecolumn chromatography, performed on a BIOCAD 700 (Applied Biosystems),was used to further investigate promising resins and conditions. XK16Glass columns (Amersham Pharmacia) were packed with different resinsusing constant flow packing methods with a linear velocity of 150cm/hour. Chromatography parameters were programmed using various methods(see the details in each experiment result). In general, the programscontain functional blocks of column equilibration, sample loading, lowsalt washing, linear or step gradient and high salt cleaning step. TheAAV vector particles and empty capsids eluted in various fractions weremonitored and analyzed using the techniques described below.

SDS-PAGE and Western Blotting:

Samples from column fractions were taken and were heated in SDS-PAGEloading buffer containing reducing agent (DTT), and capsid proteins wereresolved on pre-cast gradient polyacylamide gels (Novex). Silverstaining was performed using SilverXpress (Invitrogen, Calif.) accordingto the manufacturer's instructions. Similarly, Western blot analysis wasperformed following transfer of proteins resolved by SDS-PAGE tonitrocellulose membranes. A monoclonal antibody (B1, American ResearchProducts, MA) was used as the primary antibody to detect AAV capsidproteins (VP1, VP2, and VP3) by Western blot. Sheep anti-mouse IgG HRPconjugated (Promega, Wis.) was used as secondary antibody and detectedby ECL kit (Amersham, UK).

Quantitative Real-time PCR

The concentration of AAV vector genomes (vg) in column fractions wasmeasured by quantitative real time PCR (Q-PCR). Samples were diluted anddigested with DNase I to remove exogenous DNA. After inactivation of theDNase, the samples were further diluted and amplified using primers anda TaqMan™ fluorogenic probe specific for the DNA sequence between theprimers. The number of cycles required to reach a defined level offluorescence (threshold cycle, Ct)was measured for each sample on anApplied Biosystems Prsim 7700 Sequence Detection System. Plasmid DNAcontaining identical sequences to that contained in the AAV vector wasemployed to generate a standard curve in the Q-PCR reaction. The Ctvalues obtained from the samples were used to determine vector genometiter by normalizing it to the Ct value of the plasmid standard curve.

EXAMPLE 1 Binding Properties of Empty Capsids and AAV Vector Particleswith Packaged Genomes

To test if AAV vector particles bound to anion exchange columns, severalresins were screened using a high-throughput manifold technique. Thecell lysate of the 293 cell culture containing AAV vector particles andempty capsids was diluted using 20 mM sodium phosphate buffer to reducethe NaCl concentration to about 50 mM and loaded on each column. Thecolumns were then washed and eluted following the procedure describedabove. The collected samples were subjected to Western Blot to detectthe AAV particles distribution in the fractions. As shown in FIGS. 1Aand 1B, the AAV capsid protein-specific monoclonal antibody detectedproteins in high salt (1M NaCl) elution fractions only (FIG. 1, lane 3,5, 7 and 9). This data clearly indicated the both AAV vector particlesand empty capsids bound to all of the anionic exchange resins (both weakand strong exchangers) screened.

EXAMPLE 2 Charge Differences between Empty Capsids and AAV VectorParticles

AAV empty capsids and AAV vector particles were separated using CsClgradient centrifugation. The empty capsids resulted in a visible band ata lower salt density (about 1.3 gm/cc) and the AAV vector particlesdistributed as a visible band at a higher salt density (1.38-1.41gm/cc). The empty capsids and AAV vector particles were prepared asdescribed above and loaded on columns separately. Three resins, QSEPHAROSE, POROS 50HQ and UNOSPHERE Q, were used for columnchromatography runs which were performed using the same programs tocompare the elution profiles between empty and full particles. AAV emptycapsids eluted at a lower salt concentration (eluted in earlierfractions) as compared with the AAV vector particles (eluted in laterfractions); this phenomenon was observed for all three anionic resinstested. In the case of the UNOSPHERE Q resin, the empty capsids wereeluted in fraction 10 in a salt gradient from 0 to 500 mM NaCl, whilethe AAV vector particles eluted in fraction 11; in the case of the QSEPHAROSE column, the empty capsids were eluted in fractions 11, 12 and13 while the AAV vector particles were eluted in fractions of 13, 14 and15. The same phenomenon was observed with the POROS 50HQ column when amore shallow gradient of sodium acetate concentration was used; theempty capsids were eluted in fractions 30 to 36, while the AAV vectorparticles eluted in fractions 36 to 40. These data clearly indicate thatthere are fundamental charge differences between AAV empty capsids andthe AAV vector particles and that the two populations may be separatedusing these properties by column chromatographic techniques. Confirmingthe separation of empty capsids and AAV vector particles by theseresins, the ratio of the UV absorption at 260 nm/280 nm graduallyincreased in eluted fractions, consistent with differential elution(empty capsids, which have a lower 260/280 ratio because they lack DNA,eluting first).

EXAMPLE 3 Effect of Resins and Salts on the Elution Efficiency andResolution Between Empty Capsids and AAV Vector Particles

The resins screened in this process were all anionic exchange resins,but did exhibit differences (charge density, bead size and composition,etc.). The efficiency in binding and separating empty capsids and AAVvector particles varied from resin to resin. The empty capsids elutedusing 110 mM NaCl in the Q SEPHAROSE column. In contrast, approximately65 mM NaCl was sufficient to elute the empty capsids using the UNOSPHEREcolumn, and in the case of the POROS HQ column, approximately 130 mMNaCl was sufficient to elute the empty capsids.

Several different salts were also used to in the studies of elutionprofiles. Sodium chloride, sodium acetate, potassium acetate, ammoniumacetate and sodium citrate were subjected to side-by-side comparisons.Using the same program to perform the column chromatography runs, emptycapsid elution was delayed for several fractions when sodium acetate orammonium acetate were used in comparison with sodium chloride,suggesting that different salts have different properties in the anioniccolumn chromatography and these properties may be used to enhanceresolution in separation of empty capsid from the AAV vector particles.

EXAMPLE 4 Defining Elution Conditions for Empty Capsids

Based on the experimental data discussed above, the POROS 50HQ resin wasused to develop a column chromatography-based technique to separateempty capsids from AAV vector particles. Empty capsids prepared usingCsCl centrifugation as described above were loaded on a column packedwith POROS 50HQ resin. In separate runs, following low salt (50 mM NaCl)initial washing steps, the column was washed with either 150 mM sodiumacetate, 160 mM sodium acetate or 170 mM sodium acetate. The emptycapsids were eluted most efficiently when 170 mM NaAc was used. In aseparate chromatography run using the same salt elution profile,purified AAV vector particles remained on the column and were theneluted when a linear salt concentration using higher concentration ofsodium chloride was applied.

EXAMPLE 5 Separation of Empty Capsids from AAV Vector Particles UsingAnion Exchange Chromatography

To demonstrate that empty capsids and AAV vector particles could beseparated using column chromatography techniques, purified empty capsidsand AAV vector particles were mixed together to generate samplescontaining both empty capsids and AAV vector particles. 3 ml of emptycapsids with a concentration of 9E+13 particle/ml were mixed with 2 mlof full particles with the concentration of 7E+12 vector genomes(vg)/ml. The materials were further diluted 3-fold using 20 mM sodiumphosphate buffer at pH 7.4. 14 ml of this diluted material was loaded onto a POROS 50HQ column with a 20 mL bed volume. The columnchromatography elution profile described above was used, and the dataindicated that the empty capsids were eluted in the salt condition of170 mM sodium acetate while the AAV vector particles remained on thecolumn and were then eluted using a linear gradient of NaCl salt. The UVabsorption pattern (260:280 nm ratio) of material eluted from the columnover the course of the separation was fully consistent with and furtherdemonstrated the effective separation of the empty capsids from the AAVvector particles. The various fractions eluted from the column werefurther analyzed using capsid ELISA, Q-PCR, SDS-PAGE and OD analysis.All of the data obtained indicated that the separation of the emptycapsids from AAV vector particles was complete and that the ratio ofparticles to vector genomes in the AAV vector particle fraction was 1:1.Thus, the AAV vector particles were completely separated from the emptycapsids, despite starting material (loaded on the column) containingapproximately 16-fold more empty capsids than vector particles (FIG. 2).

EXAMPLE 6 Effect of pH on Separation of Empty Capsids from AAV VectorParticles Using Anion Exchange Chromatography

To develop a complete column-based purification process to remove emptycapsids, virions recovered from a cation exchange column (POROS HS)containing both empty capsids and AAV vector particles were applied tothe POROS 50HQ column. When a similar method to separate empty capsidsand AAV vector particles from CsCl purified samples was used, a clearseparation as observed for the CsCl-gradient pre-purified and mixedsamples was not obtained, although the two particle types remaineddistinguishable i.e. partial but incomplete separation of empty capsidsand AAV vector particles was observed. Several parameters that mayaffect the resolution of column chromatography were tested to optimizethe separation efficiency. Among parameters tested, it was found that pHenhanced the separation resolution significantly. Buffers with pH 7.4,pH 8.0, pH 8.5 and pH 9.0 were tested. The empty AAV capsids wereselectively removed in the early fractions from the column when higherpHs, such as pH 9.0, were used in the process, while the AAV vectorparticles eluted in a higher salt concentration in the later fractions.Using pH 9.0, the empty capsids were removed as a single peak with theUV absorption signature pattern of higher UV 280 signal and lower UV 260signal. In contrast, the second peak contained AAV vector particles andshowed the opposite UV absorption pattern, UV 260 dominating UV 280,than expected for the DNA containing full AAV vector particle. Theobservation was further confirmed by Q-PCR and SDS-PAGE assays,indicating that the second peak contained AAV vector particles and thefirst peak empty capsids.

EXAMPLE 7 Procedure to Separate AAV Empty Capsids from AAV VectorParticles Using Anion Exchange Column Chromatography

Based on the observation described above, a procedure was designed toseparate AAV empty capsids from the AAV vector particles using aTris-based buffer at pH 8.5 and a POROS 50HQ column resin in an XK-16glass column (Amersham Bioscience, Piscataway, N.J.). The materialseluted in early segments contained empty capsids and later segments AAVvector particles as indicated by the UV absorption patterns. This wasfurther confirmed by Q-PCR analysis. The vectors that eluted at thehigher salt concentration were analyzed on SDS-PAGE to determine purity.Vector genomes were determined by Q-PCR and replicate samples wereloaded on SDS-PAGE and stained by silver staining. To serve as acontrol, rAAV virions containing AAV vector particles only were purifiedusing CsCl centrifugation techniques and the same vector genomes wererun on the SDS-PAGE gels.

Using these techniques, approximately 60% of the vector was recovered.The vector recovered was essentially free of empty capsids (<10 of theparticles were empty capsids) based on the SDS-PAGE silver staininganalysis (FIG. 3). As shown in FIG. 3, there were no significantdifferences in the band density (corresponding to total protein) betweenthe empty capsid free vector control (lane 1) and the vector containedin eluted fractions from the column (lanes 2-5), when all lanes wereloaded with an equal number of vector genomes.

EXAMPLE 8 Separation of Empty Capsids from AAV Vector Particles UsingCation Exchange Column Chromatography

Cation exchange column chromatography was also investigated for itspotential to separate empty capsids from AAV vector particles. The AAVpreparation from the first cation exchange column (described in thematerials and methods section) containing both empty capsids and AAVvector particles, was applied to a POROS HS resin. The column was firstequilibrated with 20 mM sodium phosphate buffer containing 200 mM NaClat pH 7.4. The AAV preparation containing both AAV vector particles andempty capsids obtained from the first cation exchange column was dilutedusing 20 mM sodium phosphate, pH 7.4 to reduce the salt concentration toapproximately 200 mM NaCl. This material was then loaded onto the POROSHS column. A gradient from 500 to 700 mM ammonium acetate containing 10mM Tris at pH 8.5 was then applied to the column. Using this gradientelution protocol, the AAV vector particles eluted first, and wereefficiently separated from the empty capsids which eluted subsequentlyat a higher ammonium acetate concentration. This contrasts to theseparation observed using the anion exchange resin (Example 7 above) inwhich the empty capsids were eluted at a lower salt concentrationrelative to the AAV vector particles.

Additional information regarding the relative binding and elutionpatterns of the empty capsids and AAV vector particles on the cationexchange column was provided by the UV absorbance of the column eluate.For example, when the cation exchange column was run at pH 8.5, thechromatograph demonstrated a UV absorption pattern with 260 nmabsorption (corresponding to AAV vector particles) dominant in the earlyfractions and 280 nm absorption (corresponding to empty capsids)dominant in the later elution fractions. As shown in FIG. 4, when thesame vector genomes were subjected to SDS-PAGE silver staining analysis,the amount of protein increased significantly from early fractions tolater fractions, indicating that early fractions contained less emptycapsids.

Based on these observations, separation of empty AAV capsids from AAVvector (DNA-containing) particles using cation exchange chromatographycan be achieved. FIG. 5 shows an SDS-PAGE gel demonstrating separation.An AAV vector preparation containing both empty capsids and AAV vectorparticles eluted from a cation exchange column (in which conditionsrequired to achieve separation were not used) were then loaded on acation exchange column and eluted with a step gradient using ammoniumacetate. Fractions containing vector genomes were identified usingQ-PCR, and aliquots of these fractions were subjected to SDS-PAGE silverstaining analysis. It is clear that the protein signals in the elutedfractions contained less protein signal per vg input relative to thematerial that was loaded onto the second column, demonstrating thatempty capsids were removed from the AAV vector particles. Shown in lane2 to 4 are the vectors eluted from three independent experiments,indicating consistently and repeatability of the observations.

Thus, methods for separating empty capsids from AAV vector particles aredescribed. Although preferred embodiments of the subject invention havebeen described in some detail, it is understood that obvious variationscan be made without departing from the spirit and the scope of theinvention as defined by the appended claims.

1. A method for purifying AAV vector particles from an AAV preparationcomprising AAV vector particles and AAV empty capsids, to provide an AAVproduct substantially free of AAV empty capsids, said method comprising:(a) providing a host cell comprising AAV vector particles; (b) lysingsaid host cell to obtain a crude cell lysate comprising AAV vectorparticles and AAV empty capsids; (c) applying said crude cell lysate toa cation exchange chromatograhphy column under conditions whereby saidAAV vector particles and said AAV empty capsids bind the column; (d)eluting said AAV vector particles and said AAV empty capsids undernon-separating conditions to provide an AAV preparation comprising AAVvector particles and AAV empty capsids; (e) applying the AAV preparationfrom (d) to an anion exchange chromatography column under conditionswhereby said AAV vector particles and said AAV empty capsids bind thecolumn; (f) adding a salt buffer to the column from (e) under conditionswhereby AAV empty capsids are eluted and AAV vector particles remainbound to the column; (g) adding a buffer with a greater saltconcentration relative to the buffer added in (f) to the column from (f)under conditions whereby AAV vector particles are eluted; (h) collectingeluted fractions from (g) that comprise AAV vector particles to providean AAV product substantially free of AAV empty capsids.
 2. The method ofclaim 1, further comprising: (i) applying the AAV preparation from (h)to a second anion exchange chromatography column under conditionswhereby said AAV vector particles and said AAV empty capsids, ifpresent, bind the column; (j) adding a salt buffer to the column from(i) under conditions whereby AAV empty capsids are eluted and AAV vectorparticles remain bound to the column; (k) adding a buffer with a greatersalt concentration relative to the buffer added in (j) to the columnfrom (j) under conditions whereby AAV vector particles are eluted; (l)collecting eluted fractions from (k) that comprise AAV vector particlesto provide an AAV product substantially free of AAV empty capsids. 3.The method of claim 1, further comprising: (i) applying the AAVpreparation from (h) to a second cation exchange chromatography columnunder conditions whereby said AAV vector particles and said AAV emptycapsids bind the column; (j) adding a salt buffer to the column from (i)under conditions whereby AAV vector particles are eluted and AAV emptycapsids remain bound to the column; and (k) collecting eluted fractionsfrom (j) that comprise AAV vector particles to provide an AAV productsubstantially free of AAV empty capsids.
 4. The method of claim 1,wherein AAV vector particles are present in said AAV product in anamount of at least 75%.
 5. The method of claim 1, wherein AAV vectorparticles are present in said AAV product in an amount of at least 85%.6. The method of claim 1, wherein AAV vector particles are present insaid AAV product in an amount of at least 90%.
 7. The method of claim 1,wherein said AAV vector particles are from AAV-2.
 8. The method of claim1, wherein said AAV vector particles are from AAV-5.
 9. A method forpurifying AAV vector particles from an AAV preparation comprising AAVvector particles and AAV empty capsids, to provide an AAV productsubstantially free of AAV empty capsids, said method comprising: (a)providing a host cell comprising AAV vector particles; (b) lysing saidhost cell to obtain a crude cell lysate comprising AAV vector particlesand AAV empty capsids; (c) clarifying said crude cell lysate to providea clarified cell lysate; (d) applying said clarified cell lysate to afirst cation exchange chromatograhphy column comprising a matrix withthe functional ligand R—SO₃—, under conditions whereby said AAV vectorparticles and said AAV empty capsids bind the column; (e) eluting saidAAV vector particles and said AAV empty capsids under non-separatingconditions to provide an AAV preparation comprising AAV vector particlesand AAV empty capsids; (f) applying the AAV preparation from (e) to asecond cation exchange chromatography column under conditions wherebysaid AAV vector particles and said AAV empty capsids bind the column;(g) adding a low salt buffer to the column from (f) under conditionswhereby AAV vector particles are eluted and AAV empty capsids remainbound to the column; (h) collecting eluted fractions from (g) thatcomprise AAV vector particles; (i) applying the fractions from step (h)to an anion exchange chromatography column under conditions whereby saidAAV vector particles and AAV empty capsids, if present, bind the column;(j) adding a low salt buffer to the column from (i) under conditionswhereby AAV empty capsids are eluted and AAV vector particles remainbound to the column; (k) adding a high salt buffer to the column from(j) under conditions whereby AAV vector particles are eluted; and (l)collecting eluted fractions from (k) that comprise AAV vector particlesto provide an AAV product substantially free of AAV empty capsids. 10.The method of claim 9, wherein AAV vector particles are present in saidAAV product in an amount of at least 75%.
 11. The method of claim 9,wherein AAV vector particles are present in said AAV product in anamount of at least 85%.
 12. The method of claim 9, wherein AAV vectorparticles are present in said AAV product in an amount of at least 90%.13. The method of claim 9, wherein said AAV vector particles are derivedfrom AAV-2.
 14. The method of claim 9, wherein said AAV vector particlesare derived from AAV-5.